[go: up one dir, main page]

WO2024232423A1 - Procédé de mesure de protéoglycane - Google Patents

Procédé de mesure de protéoglycane Download PDF

Info

Publication number
WO2024232423A1
WO2024232423A1 PCT/JP2024/017331 JP2024017331W WO2024232423A1 WO 2024232423 A1 WO2024232423 A1 WO 2024232423A1 JP 2024017331 W JP2024017331 W JP 2024017331W WO 2024232423 A1 WO2024232423 A1 WO 2024232423A1
Authority
WO
WIPO (PCT)
Prior art keywords
proteoglycan
domain
measurement method
terminal domain
terminus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2024/017331
Other languages
English (en)
Japanese (ja)
Inventor
晃明 桝谷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ichimaru Pharcos Co Ltd
Original Assignee
Ichimaru Pharcos Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ichimaru Pharcos Co Ltd filed Critical Ichimaru Pharcos Co Ltd
Priority to JP2024543043A priority Critical patent/JP7626511B1/ja
Publication of WO2024232423A1 publication Critical patent/WO2024232423A1/fr
Anticipated expiration legal-status Critical
Pending legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • This disclosure relates to a method for measuring proteoglycans.
  • Proteoglycans are molecules that make up the extracellular matrix along with collagen, hyaluronic acid, etc.
  • purification of proteoglycans by extraction using guanidine hydrochloride or acetic acid has been reported (Patent Document 1 and Non-Patent Document 1).
  • proteoglycan exists in the body bound to hyaluronic acid, etc. Therefore, proteoglycan capable of binding to hyaluronic acid, etc. may be used in regenerative medicine.
  • the proteoglycan may be extracted in a state in which the C-terminal domain (hereinafter also referred to as the "C-terminus") and/or the N-terminal domain (hereinafter also referred to as the "N-terminus”) of the proteoglycan is cleaved. For this reason, it is necessary to evaluate the state of the proteoglycan, more specifically, the presence or absence of the C-terminus and/or the N-terminus of the proteoglycan.
  • the present disclosure therefore aims to provide, for example, a method for measuring proteoglycans that can detect proteoglycans whose C-terminus and/or N-terminus are maintained.
  • the method for measuring proteoglycan of the present disclosure comprises a cleavage step of cleaving disulfide bonds of the proteoglycan; a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; and detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence.
  • the present disclosure can provide, for example, a method for measuring proteoglycans that can detect proteoglycans with their C-terminus and/or N-terminus maintained.
  • FIG. 1 shows the main disaccharide structures of each chondroitin sulfate.
  • FIG. 2 shows the structures of various proteoglycans.
  • FIG. 3 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 1.
  • FIG. 4 is a photograph showing the results of detection of the N-terminal domain in proteoglycan in Example 1.
  • FIG. 5 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 1.
  • FIG. 6 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 2.
  • FIG. 7 is a photograph showing the results of decorin detection in Example 3.
  • proteoglycan refers to a molecule (glycoprotein) in which a protein (core protein) and glycosaminoglycan (GAG, also called “polysaccharide” or “sugar chain”) are covalently bonded.
  • the proteoglycan exists as an extracellular matrix in, for example, skin, organs, cartilage, and the like.
  • the glycosaminoglycan is generally known as a sugar chain having a long chain structure without a branched structure. Examples of the proteoglycan include the following.
  • Aggrecan family also called the lectican family or hyalectan family
  • Aggrecan Aggrecan
  • versican neurocan
  • neurocan neurocan
  • brevican etc.
  • Small Leucine Rich Proteoglycans (SLRPs) family biglycan, decorin, fibromodulin, lumican, PG-Lb (epiphycan), keratocan, mimecan, etc.
  • SLRPs Small Leucine Rich Proteoglycans
  • basement membrane proteoglycans perlecan, agrin, permacan, etc.
  • -Others Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc.
  • the proteoglycans can also be classified into, for example, chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, and keratan sulfate proteoglycans depending on the type of GAG bound to the protein.
  • GAG examples include chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate.
  • chondroitin examples include O-type glycans with a disaccharide structure of glucuronic acid and acetylgalactosamine as the main disaccharide structure, and iO-type glycans with a disaccharide structure of iduronic acid and acetylgalactosamine as the main structure (hereinafter, also referred to as "chondroitin sulfate O” and "chondroitin sulfate iO", respectively).
  • the chondroitin sulfate (CS) has a structure in which a sulfate group is added to a glycan in which the disaccharides of glucuronic acid and acetylgalactosamine are repeated.
  • the chondroitin sulfate may be, for example, chondroitin sulfate A (A type) whose disaccharide structure is mainly composed of glucuronic acid and acetylgalactosamine 4-sulfate, or chondroitin sulfate C (C type) whose disaccharide structure is mainly composed of glucuronic acid and acetylgalactosamine 6-sulfate.
  • the dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which disaccharides of iduronic acid and acetylgalactosamine are repeated.
  • the dermatan sulfate may be, for example, chondroitin sulfate iA (iA type) whose disaccharide structure is mainly composed of iduronic acid and acetylgalactosamine 4-sulfate, or chondroitin sulfate iC (iC type) whose disaccharide structure is mainly composed of iduronic acid and acetylgalactosamine 6-sulfate.
  • Each chondroitin sulfate may have, for example, the disaccharide structure shown in FIG. 1 as its main disaccharide structure.
  • the sulfate group (sulfo group) is bonded to a hydrogen atom, but the present disclosure is not limited to this.
  • the sulfate group of the GAG may be ionized by eliminating a hydrogen atom, or may form a salt.
  • domain refers to a region in a protein that is organized in terms of three-dimensional structure or function.
  • Figure 2 shows an example of a structure that includes the domains of aggrecan, versican, neurocan, brevican, and decorin.
  • N-terminal domain refers to, for example, the G1 domain, IGD domain (interglobular domain), G2 domain, and/or KS-rich domain of a proteoglycan when the proteoglycan is a member of the lectican family such as aggrecan.
  • the "N-terminal domain” refers to, for example, the domain on the N-terminal side of the leucine-rich repeat domain of a proteoglycan when the proteoglycan is a small leucine-rich proteoglycan such as decorin.
  • the G1 domain is known to bind to hyaluronic acid, link protein, and the like.
  • the N-terminal domain has chondroitin sulfate or dermatan sulfate in the side chain and has four cysteine residues.
  • C-terminal domain refers to the C-terminal domain including the G3 domain of a proteoglycan, for example, when the proteoglycan is a member of the lectican family such as aggrecan.
  • the "C-terminal domain” refers to the domain C-terminal to the leucine-rich repeat domain of a proteoglycan, for example, when the proteoglycan is a small leucine-rich proteoglycan such as decorin.
  • the G3 domain is known to bind to tenascin and the like. When the proteoglycan is decorin, the C-terminal domain has two cysteine residues.
  • intermediate domain refers to a domain between the N-terminal domain and the C-terminal domain of a proteoglycan, for example, when the proteoglycan is a member of the lectican family, and which contains at least one glycosaminoglycan binding domain (GAG).
  • intermediate domain refers to a domain between the N-terminal domain and the C-terminal domain of a proteoglycan, for example, when the proteoglycan is a small leucine-rich proteoglycan such as decorin, and which contains a leucine-rich repeat.
  • alkaline refers to an aqueous solution with a pH greater than 7. As used herein, when the pH of the aqueous solution in question is less than 7, 7, and greater than 7, the aqueous solution is classified as acidic, neutral, and alkaline, respectively.
  • S-S bond refers to a bond between sulfur atoms formed between two thiol groups (-SH).
  • reducing agent means a substance that can reduce other substances (can donate electrons to other substances) in an oxidation-reduction reaction.
  • modification refers to chemical modification of specific functional groups such as amino acid residues that make up a protein.
  • alkylation refers to a chemical reaction in which a hydrogen atom constituting an organic compound is replaced with an alkyl group (C n H 2n+1 ). A substance that causes the alkylation is called an alkylating agent.
  • purification means identifying, separating, recovering from a component in its natural state, being identified and separated, and/or being recovered from a component in its natural state.
  • the “purification” can be carried out, for example, by obtaining at least one purification step.
  • the purification can also be referred to as isolation.
  • extraction means taking out a specific component from a subject and/or a state in which a specific component has been taken out from a subject.
  • the extraction can be carried out, for example, using an extracting liquid such as a solvent.
  • the "extraction” can be carried out, for example, by obtaining at least one extraction process.
  • a substance containing a component taken out from the subject is called an extract.
  • a liquid containing a component taken out from the subject is called an extracting liquid.
  • the extract and the extracting liquid may contain, for example, the proteoglycan.
  • the extract or the extracting liquid may contain, for example, components other than the proteoglycan derived from the animal tissue used for the extraction (other components). Examples of the other components include lipids, nucleic acids, proteins, etc.
  • the present disclosure provides a method for measuring proteoglycan.
  • the method for measuring proteoglycan includes a cleavage step of cleaving disulfide bonds of proteoglycan, a modification step of modifying thiol groups of the obtained proteoglycan under alkaline conditions, and a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence.
  • proteoglycan with the C-terminus and/or N-terminus maintained can be detected.
  • the N-terminus and C-terminus of the proteoglycan can be measured simultaneously, so that, for example, the number of proteoglycan samples used for measurement can be reduced.
  • proteoglycans are extracted with their C-terminus and/or N-terminus maintained or cleaved, depending on the type of extraction solution, etc.
  • the inventors have found that the C-terminus and/or N-terminus of proteoglycans can be detected by fluorescence detection by cleaving the disulfide bonds of purified proteoglycans and modifying the thiol groups of the proteoglycans under alkaline conditions to inhibit the formation of complexes between the proteoglycans and hyaluronic acid, etc., and have established the present disclosure.
  • the measurement method of the present disclosure makes it possible to detect proteoglycans with their C-terminus and/or N-terminus maintained.
  • disulfide bonds in the proteoglycan are cleaved. This makes it possible, for example, to destroy the three-dimensional structure of the proteoglycan, and, in the case where the proteoglycan has a C-terminus and/or an N-terminus, to dissolve the formation of a complex between the proteoglycan and the hyaluronic acid or the like. Therefore, in the case where the proteoglycan has a C-terminus and/or an N-terminus, the cleavage process makes the C-terminus and/or the N-terminus of the proteoglycan exposed, i.e., rendered detectable.
  • the cleavage step can be carried out, for example, by a reaction system (cleavage system) containing the proteoglycan and a component capable of cleaving the disulfide bond of the proteoglycan.
  • the cleavage system is, for example, a liquid system such as an aqueous solution.
  • the proteoglycan is contacted with a component capable of cleaving a disulfide bond to cleave the disulfide bond present in the proteoglycan.
  • the contact between the proteoglycan and the component capable of cleaving the disulfide bond can be carried out, for example, by a known contact method between a solid and a liquid, and specifically, can be carried out by mixing the proteoglycan with a solution containing a component capable of cleaving the disulfide bond.
  • the component capable of cleaving the disulfide bond can be, for example, a reducing agent.
  • the reducing agent is used in the cleavage step, the cleavage can also be referred to as reduction to a thiol group by the reducing agent.
  • the reducing agent is not particularly limited, and is, for example, a compound capable of reducing a disulfide bond to a free thiol group.
  • Examples of the reducing agent include dithiothreitol (DL-dithiothreitol, DTT), ⁇ -mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), glutathione, etc.
  • the cleavage conditions (temperature, time, etc.) in the cleavage step are not particularly limited and can be set, for example, under conditions under which the disulfide bond is sufficiently cleaved into the thiol group.
  • the cleavage conditions can also be set appropriately depending on, for example, the type of the reducing agent, etc.
  • the reaction temperature in the cleavage is, for example, preferably 45 to 100°C, more preferably 50 to 60°C.
  • the reaction time in the cleavage is, for example, preferably 3 minutes to 6 hours, more preferably 5 minutes to 3 hours.
  • the proteoglycan may be, for example, an animal-derived proteoglycan.
  • the animal is not particularly limited, and may be, for example, fish such as salmonids, such as chum salmon and Atlantic salmon, and flatfish, such as platypus; avian animals (birds), such as chickens; and mammalian animals (mammals), such as pigs.
  • the animal is preferably salmon, chicken, pig, or flatfish.
  • the proteoglycan may be, for example, a proteoglycan derived from a tissue that contains proteoglycan.
  • tissue include epithelial tissue such as skin; cartilage tissue such as cartilage; digestive organs; circulatory organs; respiratory organs; and placenta.
  • Specific examples of the tissue include cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.
  • the sugar chain of the proteoglycan may be, for example, chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate.
  • the sugar chain is preferably chondroitin sulfate.
  • the proteoglycan used in the measurement method of the present disclosure may be a self-prepared proteoglycan or a commercially available proteoglycan.
  • the measurement method of the present disclosure may include, for example, a step of extracting and/or purifying proteoglycan from an animal or animal tissue prior to the cutting step.
  • the proteoglycan may be a proteoglycan purified by acetic acid extraction or a proteoglycan purified by guanidine extraction.
  • the proteoglycan can be purified with reference to the method of Japanese Patent No. 3731150.
  • the proteoglycan can be purified with reference to the methods of References 1 and 2 below.
  • Reference 1 Kakizaki I, Tatara Y, Majima M, Kato Y, Endo M. Identification of proteoglycan from salmon nasal cartilage. Arch Biochem Biophys. 2011 Feb 1;506(1):58-65. doi: 10.1016/j.abb.2010.10.025. Epub 2010 Nov 5.
  • Reference 2 Kakizaki I, Mineta T, Sasaki M, Tatara Y, Makino E, Kato Y. Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan. Carbohydr Polym. 2014 Mar 15;103:538-49. doi: 10.1016/j.carbpol.2013.12.083. Epub 2014 Jan 7. Erratum in: Carbohydr Polym. 2015 Jan 22;115:805.
  • the molecular weight of the proteoglycan may be measured before the cutting step.
  • the molecular weight of the proteoglycan means the peak top molecular weight measured by GPC (Gel Permeation Chromatography).
  • the molecular weight of the proteoglycan can be measured using the GPC method or the like.
  • the GPC method can be performed, for example, under the following conditions, and the standard sample (molecular weight marker, pullulan) can be individually injected into an HPLC system to calculate the molecular weight calibration curve.
  • STD P-800 Mp: 739,000, Mw/Mn: 1.24 STD P-400: Mp: 348,000, Mw/Mn: 1.33 STD P-200: Mp: 216,000, Mw/Mn: 1.22 STD P-100: Mp: 107,000, Mw/Mn: 1.12 STD P-50: Mp: 49,400, Mw/Mn: 1.08 STD P-20: Mp: 22,000, Mw/Mn: 1.08 STD P-10: Mp: 9,800, Mw/Mn: 1.07 STD P-5: Mp: 6,300, Mw/Mn: 1.09
  • the peak top molecular weight of the proteoglycan subjected to the measurement is, for example, 300,000 to 1,300,000, or 400,000 to 1,200,000.
  • the modification step the free thiol groups of the proteoglycan obtained in the cleavage step are modified under alkaline conditions.
  • the modification step for example, the free thiol groups of the proteoglycan are modified, and the reformation of disulfide bonds between the thiol groups can be inhibited. Therefore, in the case where the proteoglycan has a C-terminus and/or an N-terminus, the modification step can maintain the C-terminus and/or the N-terminus of the proteoglycan in an exposed state, i.e., in a detectable state.
  • the modification step can maintain the C-terminus and/or the N-terminus of the proteoglycan in a detectable state, and therefore the C-terminus and/or the N-terminus can be detected in the detection step described below.
  • the modification step can be carried out, for example, in a reaction system (modification system) containing the proteoglycan obtained in the cleavage step and a modifying agent used for the modification.
  • the reaction system is, for example, a liquid system such as an aqueous solution.
  • the reaction system can be prepared, for example, by mixing the proteoglycan and the modifying agent.
  • the modification step is preferably carried out under alkaline conditions.
  • the pH in the modification step (modification pH) is, for example, preferably pH 7.5 to pH 8.5, and more preferably pH 7.8 to pH 8.2.
  • the thiol groups can be suitably modified, and the C-terminus and/or N-terminus of the proteoglycan can be suitably detected.
  • the pH of the reaction system can be adjusted using, for example, an alkali such as sodium hydroxide, potassium hydroxide, potassium hydroxide, sodium bicarbonate, or sodium borate; or an acid such as hydrochloric acid, sulfuric acid, nitric acid, citric acid, or phosphoric acid.
  • the modification may be, for example, a chemical modification such as alkylation, glutathionylation, sulfonation, sulfenation or sulfination, or nitrosation (nitrosylation).
  • the modification may be carried out, for example, by using a modifying agent (reactant) containing a modifying group imparted by the modification.
  • the alkylation may be carried out, for example, by using an alkylating agent.
  • the alkylating agent is, for example, a compound capable of reacting with the thiol group to alkylate the thiol group.
  • the alkylating agent may be, for example, an organic iodide salt such as iodoacetamide, methyl iodide, or iodoacetic acid, or an alkylene oxide such as ethylene oxide.
  • the alkylating agent may be, for example, one type, or a combination of multiple types.
  • the sulfenation or sulfination may be carried out, for example, by using a compound capable of oxidizing the thiol group.
  • the compound capable of oxidizing the thiol group may be, for example, an oxidizing agent.
  • the nitrosation can be carried out, for example, using a compound that can donate nitric oxide (NO), specifically, S-nitrosoglutathione, etc.
  • the modification conditions (temperature, time, etc.) in the modification step are not particularly limited and can be set to conditions under which the thiol group is sufficiently modified, for example.
  • the reaction temperature in the modification is, for example, preferably 15 to 40°C.
  • the reaction time in the modification is, for example, preferably 5 minutes or more, and more preferably 5 minutes to 16 hours.
  • the modification may include, for example, components other than the modifying agent.
  • the other components include a buffer such as a buffer, a pH adjusting agent, etc.
  • the measurement method of the present disclosure may optionally include a solid-phasing step of solidifying the proteoglycan on a carrier such as a substrate after the modification step and prior to the detection step.
  • the solid-phasing method can be carried out, for example, by a known method for solidifying proteins, specifically, by a covalent bond method, an ionic bond method, a physical adsorption method, a hydrophobic interaction method, or the like.
  • the carrier examples include plates such as microwell plates, microarray substrates, membranes (e.g., polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, polystyrene nonwoven fabrics, etc.), and low-fluorescence carriers (e.g., low-fluorescence plates, low-fluorescence membranes, etc.).
  • plates such as microwell plates, microarray substrates, membranes (e.g., polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, polystyrene nonwoven fabrics, etc.), and low-fluorescence carriers (e.g., low-fluorescence plates, low-fluorescence membranes, etc.).
  • PVDF polyvinylidene fluoride
  • nitrocellulose membranes nitrocellulose membranes
  • polystyrene nonwoven fabrics etc.
  • low-fluorescence carriers e.g., low-fluorescence plates, low-fluorescence membranes, etc.
  • blocking may be performed on the carrier so that components such as antibodies used in the detection step described below do not bind nonspecifically.
  • blocking agents used in the blocking step include albumin such as bovine serum albumin (BSA), skim milk, commercially available blocking reagents, etc.
  • the detection step at least one of the C-terminal domain and the N-terminal domain of the modified proteoglycan is detected by fluorescence.
  • the detection method can be carried out by contacting the modified proteoglycan with a probe and detecting the probe bound to the proteoglycan by fluorescence.
  • a substrate or the like having the probe or proteoglycan immobilized thereon may be used.
  • either the C-terminal domain or the N-terminal domain of the modified proteoglycan may be detected, or both may be detected. In the latter case, it is preferable that, for example, the C-terminal domain and the N-terminal domain of the proteoglycan are detected simultaneously by fluorescence in the detection step.
  • the N-terminal domain of the proteoglycan to be detected can be, for example, the G1 domain, the IGD domain, or the G2 domain, and is preferably the G1 domain. In the detection, for example, one, two, or three of the N-terminal domains can be detected.
  • the C-terminal domain of the proteoglycan to be detected can be, for example, the G3 domain.
  • the detection can be performed, for example, by detecting the fluorescence of the target component, and specifically, by an immunological assay.
  • the immunological assay include a direct method for detecting the binding between a probe and a proteoglycan; an indirect method for binding a first probe to a proteoglycan and then detecting the first probe bound to the proteoglycan using a second probe; a sandwich method for binding a first probe to a proteoglycan and then detecting the proteoglycan bound to the first probe using a second probe; a Western blotting method, an immunoprecipitation method, and an immunohistochemical staining method.
  • the immunological assay may be a quantitative method or a qualitative method.
  • the probe examples include target-specific proteins such as polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments; target-specific nucleic acid molecules such as aptamers; etc.
  • the antibody may be an antibody of any isotype among immunoglobulins selected from IgG, IgM, IgA, IgD, IgE, and IgY.
  • Examples of the antibody fragment include antibody fragments such as F(ab') 2 , Fab', Fab, and Fv.
  • the probe may be, for example, labeled.
  • the antibody may be, for example, an antibody labeled for detection.
  • the label include a fluorescent dye label, an enzyme label, and a radioactive label.
  • the probe is preferably, for example, a fluorescently labeled probe.
  • the second probe is preferably, for example, a fluorescently labeled probe.
  • the fluorescence detection can be performed, for example, using a fluorescence detector.
  • Example 1 The measurement method of the present disclosure made it possible to detect the C-terminus and N-terminus of proteoglycan.
  • proteoglycan fraction was collected. After the collection, the proteoglycan fraction was freeze-dried to obtain proteoglycan by guanidine extraction.
  • a primary antibody (anti-aggrecan G3 domain antibody, Anti-Aggrecan Antibody clone 1-C-6, MABT84, manufactured by Merck) diluted 1/1000 was added, and the primary reaction was carried out under the conditions of 4° C. and 16 hours. After the primary reaction, the membrane was washed with TBS (Tris buffer) containing 0.05% Tween-20. After the washing, a secondary antibody (goat anti-mouse IgG StarBright Blue 700, 12004159, BioRad) diluted 1/2500 was added, and a secondary reaction was carried out at 25°C for 1 hour.
  • TBS Tris buffer
  • a secondary antibody goat anti-mouse IgG StarBright Blue 700, 12004159, BioRad
  • Figure 3 is a photograph showing the detection results of the C-terminal domain in proteoglycan.
  • the upper part of Figure 3 shows the results when disulfide bonds were cleaved and alkylated, and the lower part of Figure 3 shows the results when disulfide bonds were not cleaved and alkylated.
  • "light” indicates 10 ⁇ l of each proteoglycan
  • "medium” indicates 50 ⁇ l of each proteoglycan
  • “high” indicates 100 ⁇ l of each proteoglycan.
  • the signal of the C-terminal domain was weakly detected in all samples regardless of the extraction method.
  • the proteoglycan was derived from the nasal cartilage of chum salmon, the sternal cartilage of chicken, the bronchial cartilage of pig, or the fin of the Japanese flounder, 10 ⁇ l or 50 ⁇ l of the proteoglycan was used for immobilization, and the primary antibody was an anti-aggrecan IGD domain antibody (Aggrecan Antibody, DF7561, epitope: V355-V375, manufactured by Affinity Biosciences) diluted 1/1000, and the secondary antibody was a goat anti-rabbit IgG StarBright Blue 700 (12004162, manufactured by BioRad) diluted 1/2500. Detection was performed in the same manner as in Example 1 (3). These results are shown in FIG.
  • Figure 4 is a photograph showing the detection results of the N-terminal domain in proteoglycan.
  • the upper row (low concentration) of Figure 4 shows the results of 10 ⁇ l of each proteoglycan, and the lower row (high concentration) of Figure 4 shows the results of 50 ⁇ l of each proteoglycan.
  • the N-terminal domain could not be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid.
  • the N-terminal domain could be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with guanidine, the proteoglycan derived from chicken breast cartilage, the proteoglycan derived from bronchial cartilage of pig, and the proteoglycan derived from the fin of Japanese flounder. From these results, it was found that by using the measurement method of the present invention, the N-terminus is not preserved in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, but the N-terminal domain is preserved in the proteoglycans derived from various animals extracted with guanidine.
  • Example 1(3) Detection of C-terminal domains of various proteoglycans
  • the C-terminal domains of the proteoglycans extracted with guanidine and extracted with acetic acid obtained in Example 1(1) or Example 1(2) were detected. Specifically, the detection was performed in the same manner as in Example 1(3) except that the proteoglycans used were those derived from chum salmon nasal cartilage, chicken sternal cartilage, or pig bronchial cartilage, and 10 ⁇ l or 50 ⁇ l of the proteoglycans were used for immobilization. These results are shown in FIG. 5.
  • Figure 5 is a photograph showing the detection results of the C-terminal domain in proteoglycan.
  • the upper row of Figure 5 shows the results for 10 ⁇ l of each proteoglycan, and the lower row of Figure 5 shows the results for 50 ⁇ l of each proteoglycan.
  • the C-terminal domain could be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, as well as in the proteoglycan derived from nasal cartilage of chum salmon extracted with guanidine, the proteoglycan derived from chicken breast cartilage, and the proteoglycan derived from porcine bronchial cartilage.
  • the C-terminal domain is preserved in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid and in the proteoglycans derived from various animals extracted with guanidine.
  • Example 1 Detection of Various Domains of Various Proteoglycans
  • the various domains were N-terminal domain 1 (epitope: Val20-Gly675, G1-IGD-G2 domain), N-terminal domain 2 (epitope: aa50-150, G1 domain), C-terminal domain 1 (epitope: aa2358-2481, G3 Lectin-BD domain), and C-terminal domain 2 (epitope: aa2255-2280).
  • the detection was performed in the same manner as in Example 1(3) except that an antibody capable of detecting the various domains was used as the primary antibody and an antibody capable of detecting the primary antibody was used as the secondary antibody.
  • the primary antibodies are shown in Table 1 below.
  • the primary antibody used to detect the N-terminal domain 1 was diluted 500-fold
  • the primary antibody used to detect the N-terminal domain 2 was diluted 900-fold
  • the primary antibody used to detect the C-terminal domain 1 was diluted 30-fold
  • the primary antibody used to detect the C-terminal domain 2 was diluted 100-fold.
  • the secondary antibody used was goat anti-rabbit IgG StarBright Blue 700 (12004162, BioRad) diluted 1/2500 or goat anti-mouse IgG StarBright Blue 700 (12004159, BioRad) diluted 1/2500.
  • proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid the N-terminal domain was hardly detected, and in the C-terminal domain, a part of the G3 domain was detected, but the lectin domain was not detected.
  • proteoglycan derived from nasal cartilage of chum salmon proteoglycan derived from chicken breast cartilage, and proteoglycan derived from porcine bronchial cartilage extracted with guanidine, all N-terminal domains were detected, and all G3 domains were detected in the C-terminal domain.
  • Example 2 The measurement method of the present disclosure made it possible to detect the C-terminus and N-terminus of proteoglycan.
  • the various domains are the G1 domain, the IGD domain, the G1-IGD-G2 domain, the G3 domain 1, the G3 domain 2 (epitope: aa2255-2280), and the G3 lectin domain.
  • detection was performed in the same manner as in Example 1(3) above, except that an antibody capable of detecting the various domains described above was used as the primary antibody, and an antibody capable of detecting the primary antibody was used as the secondary antibody. The results are shown in FIG.
  • the primary antibodies are shown in Table 2 below.
  • the primary antibody detecting the G1 domain was diluted 900-fold
  • the primary antibody detecting the IGD domain was diluted 1000-fold
  • the primary antibody detecting the G1-IGD-G2 domain was diluted 500-fold
  • the primary antibody detecting the G3 domain 1 was diluted 1000-fold
  • the primary antibody detecting the G3 domain 2 (epitope: aa2255-2280) was diluted 100-fold
  • the primary antibody detecting the G3 lectin domain was diluted 30-fold.
  • the secondary antibody used was goat anti-rabbit IgG StarBright Blue 700 (12004162, BioRad) diluted 1/2500 or goat anti-mouse IgG StarBright Blue 700 (12004159, BioRad) diluted 1/2500.
  • HPLC measurement conditions HPLC system: LC-10AD Column: TSKgel G5000-PWXL ⁇ 7.8mm ⁇ 300mm (Tosoh Corporation) Eluent: pH 6.8 phosphate buffer Flow rate: 0.5 ml/min Column temperature: 40°C Detector: Differential refractive index detector (RID-10A, manufactured by Shimadzu Corporation) Injection volume: 50 ⁇ l Molecular weight marker: Shodex STANDARD P-82 (pullulan)
  • Figure 6 is a photograph showing the results of detection of C-terminal domains in proteoglycans. From top to bottom, Figure 6 shows the results for the G1 domain, IGD domain, G1-IGD-G2 domain, G3 domain 1, G3 domain 2 (epitope: aa2255-2280), and G3 lectin domain.
  • Figure 6 also shows the results for BSA (1), acetic acid-extracted proteoglycan from chum salmon nasal cartilage (2) (sCSPG-AcOH), guanidine-extracted proteoglycan from chum salmon nasal cartilage (3) (sCSPG-Gdm), guanidine-extracted proteoglycan from chicken breast cartilage (4) (cCSPG), and guanidine-extracted proteoglycan from pig bronchial cartilage (5) (pCSPG).
  • the N-terminal domain was hardly detected in the acetic acid-extracted proteoglycan, and the C-terminal domain was detected in part of G3.
  • both the N-terminal domain and the C-terminal domain were detected in the acetic acid-extracted proteoglycan, the guanidine-extracted proteoglycan, cCSPG, and pCSPG.
  • the peak top molecular weights of the acetic acid-extracted proteoglycan, the guanidine-extracted proteoglycan, cCSPG, and pCSPG were 480,000, 680,000, 630,000, and 640,000, respectively.
  • GAG analysis was performed for the various proteoglycans used in Example 2(1). Specifically, GAG was purified from the various proteoglycans used in Example 2(1). 10 g of the sCSPG-Gdm, the cCSPG, or the pCSPG was treated with 80 ml of boiling water for 10 minutes. After the treatment, 100 ml of 0.5 mol/l borate buffer (pH 7.0) was added. After the addition, 0.67 g of Protease N Amano G (manufactured by Amano Enzyme Co., Ltd.) was added, and the mixture was incubated for 7 days under the condition of 55°C.
  • the column was washed stepwise with 160 ml of buffer containing 0.15, 0.5, 1.0, or 2.0 mol/l LiCl. After the washing, the carbazole-positive fraction was collected and dialyzed, and the dialyzed sample was desalted with a gel permeation column (LH-20, H 2 O, ⁇ 1.1 ⁇ 80 cm) to obtain GAG. Then, 50 ⁇ g of the GAG was diluted with 50 ⁇ g of H 2 O. After the dilution, HPLC analysis was performed using linearly coupled size exclusion columns (OHpak SB-G 6B 100 ⁇ 4.6 mm and SB-805 HQ 250 ⁇ 4.6 mm).
  • HPLC measurement conditions HPLC system: LC-10AD (Shimadzu Corporation) Columns: OHpak SB-G 6B 100 ⁇ 4.6mm and SB-805 HQ 250 ⁇ 4.6mm Eluent: 0.1 mol/l NaNO3 Flow rate: 1mL/min
  • the peak top molecular weight (Mp) of guanidine-extracted GAG (sGAG) derived from nasal cartilage of chum salmon was 84,000
  • the peak top molecular weight (Mp) of guanidine-extracted GAG (cGAG) derived from chicken breast cartilage was 77,000
  • the peak top molecular weight (Mp) of guanidine-extracted GAG (pGAG) derived from porcine bronchial cartilage was 51,000.
  • Example 2(2) Disaccharide Structure in Each Proteoglycan
  • 50 ⁇ g of each of the GAGs obtained in Example 2(2) was diluted with 50 ⁇ l H 2 O, a BSA solution (0.05 mg/5 ⁇ l), and 10 ⁇ l of a 250 mmol/l Tris-HCl (AcOH) solution (pH 8.0).
  • the resulting diluted solution was digested with chondroitinase ABC (25 mU/25 ⁇ l, Sigma Aldrich) or chondroitinase AC (25 mU/25 ⁇ l, Sigma Aldrich) at 37° C.
  • bovine proteoglycan skin sulfate proteoglycan: DSPG
  • DSPG bovine proteoglycan
  • As the raw material about 824 g of the abomasum of a bovine (Holstein) was prepared. The abomasum was washed and chopped into about 2 cm cubes while removing as much fat as possible. After the chopping, four times the amount of ethanol (3288 ml) was added, and the mixture was left to stand for 10 minutes, and this was repeated once. Then, four times the amount of ethanol (3288 ml) was added, and the mixture was left to stand overnight. After the standing, the ethanol was removed, and the mixture was air-dried.
  • Holstein bovine proteoglycan
  • the mixture was dried under reduced pressure to obtain 173 g of a defatted and dried product.
  • the proteoglycan was purified from the defatted and dried product by guanidine extraction. Specifically, 87 g of the defatted and dried product of Example 2 (1-1) was added with 350 ml of 4 M guanidine hydrochloride aqueous solution, and immersion extraction was performed at 4°C for 3 days. After the immersion extraction, 347 ml of 4 M guanidine hydrochloride aqueous solution was added, and further immersion extraction was performed at 4°C for 3 days. After the immersion extraction, suction filtration and dialysis were performed. After the dialysis, freeze-drying was performed to obtain 9.93 g of proteoglycan solids.
  • the proteoglycan was subjected to salt exchange using a neutral buffer (pH 7.5). Specifically, the salt exchange using the neutral buffer (pH 7.5) was performed by adding 60 ml of neutral buffer (pH 7.5) to the bovine stomach-derived proteoglycan. After the addition, the proteoglycan was subjected to ultrasonic disruption and allowed to stand overnight. After the standing, the proteoglycan was centrifuged to obtain a supernatant. The supernatant was subjected to dialysis and freeze-drying.
  • a neutral buffer pH 7.5
  • the salt exchange using the neutral buffer (pH 7.5) was performed by adding 60 ml of neutral buffer (pH 7.5) to the bovine stomach-derived proteoglycan. After the addition, the proteoglycan was subjected to ultrasonic disruption and allowed to stand overnight. After the standing, the proteoglycan was centrifuged to obtain a supernatant. The supernatant was subjected to dialysis
  • the PVDF membrane was washed with TBS containing 0.05% Tween-20 (Tris buffer, TBS-T). After the washing, the PVDF membrane was blocked with EveryBlot blocking buffer (BioRad) for 5 minutes. After the blocking, a primary antibody (anti-Decorin antibody, Cat No: 14667-1-AP, Proteintech) diluted 1/1000 was added, and the primary reaction was carried out at 4°C for 18 hours. After the primary reaction, the cells were washed five times with TBS-T for 5 minutes. After the washing, StarBright Blue (BioRad) diluted 1/2500 and 0.05% SDS were added, and the secondary reaction was carried out at room temperature for 1 hour.
  • TBS Tris buffer, TBS-T
  • Figure 7 is a photograph showing the results of decorin detection.
  • the left column shows the results for BSA, and the right column shows the results for bovine-derived DSPG.
  • a signal could be detected in bovine-derived DSPG.
  • a method for measuring proteoglycan comprising: A cleavage step of cleaving disulfide bonds of the proteoglycan; a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence;
  • a method for measuring proteoglycan comprising: (Appendix 2) The measurement method according to claim 1, wherein the detection step comprises simultaneously detecting the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence.
  • (Appendix 11) The method for measuring the peak top molecular weight of the proteoglycan according to any one of claims 1 to 10, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000.
  • (Appendix 12) The method for measuring the proteoglycan according to any one of claims 1 to 11, wherein the proteoglycan is an animal-derived proteoglycan.
  • (Appendix 13) The method of claim 12, wherein the proteoglycan is a proteoglycan of the lectican family.
  • (Appendix 14) The measurement method according to claim 12 or 13, wherein the proteoglycan is aggrecan.
  • the present disclosure for example, it is possible to provide a method for measuring proteoglycan capable of detecting proteoglycan with its C-terminus and/or N-terminus maintained, etc. For this reason, the present disclosure can be said to be extremely useful, for example, in the field of regenerative medicine.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La divulgation concerne un procédé de mesure d'un protéoglycane permettant de détecter un protéoglycane dans lequel l'extrémité C-terminale et/ou l'extrémité N-terminale sont maintenues.　Un procédé de mesure d'un protéoglycane selon la présente divulgation comprend : une étape de coupe pour découper une liaison disulfure dans le protéoglycane ; une étape de modification pour modifier des groupes thiol dans le protéoglycane obtenu dans des conditions alcalines ; et une étape de détection pour détecter le domaine C-terminal et/ou le domaine N-terminal du protéoglycane par fluorescence.
PCT/JP2024/017331 2023-05-11 2024-05-10 Procédé de mesure de protéoglycane Pending WO2024232423A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2024543043A JP7626511B1 (ja) 2023-05-11 2024-05-10 プロテオグリカンの測定方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2023-078729 2023-05-11
JP2023078729 2023-05-11
JP2023206830 2023-12-07
JP2023-206830 2023-12-07

Publications (1)

Publication Number Publication Date
WO2024232423A1 true WO2024232423A1 (fr) 2024-11-14

Family

ID=93430158

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2024/017331 Pending WO2024232423A1 (fr) 2023-05-11 2024-05-10 Procédé de mesure de protéoglycane

Country Status (2)

Country Link
JP (1) JP7626511B1 (fr)
WO (1) WO2024232423A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009044900A1 (fr) * 2007-10-05 2009-04-09 Hokkaido University Appareil permettant de traiter automatiquement une chaîne de sucre
JP2016160226A (ja) * 2015-03-03 2016-09-05 国立大学法人弘前大学 動物の軟骨からプロテオグリカンを調製する方法
JP2018151206A (ja) * 2017-03-10 2018-09-27 一丸ファルコス株式会社 プロテオグリカン分析方法
JP2020165937A (ja) * 2019-03-29 2020-10-08 国立大学法人弘前大学 プロテオグリカンの測定方法、評価方法および測定用キット

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009044900A1 (fr) * 2007-10-05 2009-04-09 Hokkaido University Appareil permettant de traiter automatiquement une chaîne de sucre
JP2016160226A (ja) * 2015-03-03 2016-09-05 国立大学法人弘前大学 動物の軟骨からプロテオグリカンを調製する方法
JP2018151206A (ja) * 2017-03-10 2018-09-27 一丸ファルコス株式会社 プロテオグリカン分析方法
JP2020165937A (ja) * 2019-03-29 2020-10-08 国立大学法人弘前大学 プロテオグリカンの測定方法、評価方法および測定用キット

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
ANONYMOUS: "Detection of Reductively-Alkylated Cysteines Using Protein Sequencer ", SHIMADZU; APPLICATION NEWS NO. B116, 1 November 2020 (2020-11-01), XP093235296, Retrieved from the Internet <URL:https://www.shimadzu.co.kr/sites/shimadzu.co.kr/files/pim/pim_document_file/applications/application_note/13477/b116_e.pdf> *
KAKIZAKI IKUKO, MIURA AYAKO, MINETA TAKASHI, HONG JINSEO, KATO YOJI: "Characterization of Proteoglycan and Hyaluronan in Hot Water Extract from Salmon Cartilage", JOURNAL OF APPLIED GLYCOSCIENCE, JAPANESE SOCIETY OF APPLIED GLYCOSCIENCE, JP, vol. 64, no. 4, 1 January 2017 (2017-01-01), JP , pages 83 - 90, XP093235297, ISSN: 1344-7882, DOI: 10.5458/jag.jag.JAG-2017_005 *

Also Published As

Publication number Publication date
JPWO2024232423A1 (fr) 2024-11-14
JP7626511B1 (ja) 2025-02-04

Similar Documents

Publication Publication Date Title
Maruyama Connectin, an elastic filamentous protein of striated muscle
Meyer Mucoids and glycoproteins
Rosa et al. The major tyrosine-sulfated protein of the bovine anterior pituitary is a secretory protein present in gonadotrophs, thyrotrophs, mammotrophs, and corticotrophs.
Hall et al. The apical lamina of the sea urchin embryo: Major glycoproteins associated with the hyaline layer
Hughes Membrane glycoproteins: a review of structure and function
Van den Heuvel et al. Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane
Loos Biosynthesis of the collagen-like C1q molecule and its receptor functions for Fc and polyanionic molecules on macrophages
Wolfe et al. Migration of histones from the nuclei of isolated cerebral tissues kept in cold media
Misra et al. Glomerular basement membrane in experimental nephrosis: Chemical composition
Howe The influenza virus receptor and blood group antigens of human erythrocyte stroma
JP7626511B1 (ja) プロテオグリカンの測定方法
Howe et al. Immunochemical study of hemoglobin-free human erythrocyte membranes
Armstrong A quantitative comparison of the amino acid composition of sound dentine, carious dentine and the collagenase resistant fraction of carious dentine
AU619177B2 (en) Receptor of the small rhinovirus receptor group
US4798800A (en) Process for isolating human globular domain NC1 of basal membrane collagen from human tissue
RU2621311C1 (ru) Способ получения протеогликана
Gordon et al. An improved method of preparing haptoglobin polypeptide chains using guanidine hydrochloride
Antonyuk et al. Lectin purification from fruiting bodies of brown roll-rim fungus, Paxillus involutus (Fr.) Fr., and its application in histochemistry
Aikawa et al. Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta
Merezhinskaya et al. Expression of monocarboxylate transporter 4 in human platelets, leukocytes, and tissues assessed by antibodies raised against terminal versus pre-terminal peptides
Utarabhand et al. Sialic acid-specific lectin participates in an immune response and ovarian development of the banana shrimp Fenneropenaeus merguiensis
JP2508915B2 (ja) 抗ＳＳＡ／ＲｏおよびＳＳＢ／Ｌａ抗体測定用抗原、その製造法ならびに抗ＳＳＡ／ＲｏおよびＳＳＢ／Ｌａ抗体の測定法
Lindahl Comment on the use of cetylpyridinium chloride in the isolation of connective-tissue proteoglycan
Anfinsen et al. Advances in protein chemistry
CN101805403B (zh) 原核表达Arresten蛋白纯化方法

Legal Events

Date Code Title Description
WWE Wipo information: entry into national phase

Ref document number: 2024543043

Country of ref document: JP

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24803532

Country of ref document: EP

Kind code of ref document: A1